Thermo multiskan mk3 spectrophotometer

The free radical scavenging potential of compound 6 was evaluated using the DPPH free radical scavenging assay described by Sharma and Bhat [16 (link)]. A total of 200 μL of the reaction mixture in a 96-well plate was composed of 100 μL of 0.1 mM DPPH (Sigma Aldrich, St. Louis, MO, USA) in methanol and 100 μL of different concentrations of test compound. The reaction mixture was then shaken well and incubated for 30 min in darkness at 37 °C. Meanwhile, a sample composed of 100 μL of methanol with 100 μL of 0.1 mM DPPH was taken as a control, while vitamin C (Shanxi Yishengtang Pharmaceutical Co., Ltd., Tongchuan, China) was taken as a reference material. The absorbance of reaction solution was measured at 517 nm with a plate reader (Thermo Multiskan MK3 spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA), and the percentage of scavenging capacity (%) was calculated by the following equation, where A_Control stands for the absorbance of the control and A_Treatment stands the absorbance of the treatment:
The DPPH IC₅₀ value is the concentration required to scavenge DPPH radical by 50%.

Glucose consumption was detected using a glucose assay kit according to the manufacturer's protocol. The absorbance at 570 nm was measured with a plate reader (Thermo Multiskan MK3 spectrophotometer; Thermo Fisher Scientific, Waltham, MA). Lactate levels in the culture media were determined using a lactate assay kit according to the manufacturer's protocol.
Total intracellular LDH activity was examined using an LDH activity assay kit according to the manufacturer's instructions. The results were normalized to the total protein concentration. Briefly, 2 × 10⁵ cells per well were plated in a 24-well plate for 48 h. The cells were collected, washed, and protein was extracted for measurement of LDH-A activity.
Cellular ATP was measured using a luciferin/luciferase assay kit. Cells were permeabilized prior to the addition of luciferin substrate and luciferase. Bioluminescence was assessed on an LUMATLB9507 luminometer (EG&G Berthold, Bad Wildbad, Germany) and cellular ATP content (nmol/mg) was determined using a curve for the luminescence of standard dilutions of ATP. ATP content was normalized to protein content.